| Background:The brain-gut axis is a bidirectional connection pathway between the gut and the central nervous system.Gut health and associated gut microbiota homeostasis not only participate in the gastrointestinal environment,but also affect the brain function.However,the mechanism by which gut dysfunction exacerbates brain injury is unclear.Ischemic stroke is one of the leading causes of death and disability.Therefore,exploring the intrinsic neuroprotective strategy of ischemic stroke is particularly important.Hydrogen sulfide（H2S）is the third gaseous molecule.On the one hand,H2S participates in the physiological process of the cardiovascular and cerebrovascular systems.On the other hand,H2S plays an important role in the central nervous system,intestine,kidney and other organs under pathological conditions.In this study,we established a mouse model of cerebral ischemia/reperfusion（I/R）-induced brain injury and dextran sulfate sodium（DSS）-induced colitis to explore whether the ischemic brain injury and colon inflammation promote each other and explore whether its connecting factor is H2S.The results of this study will provide ideas for exploring the neuroprotective targets of ischemic stroke.Purpose:1.To investigate therelationship between I/R-induced brain injury and dextran sulfate sodium（DSS）-induced colon inflammation in mice.2.To investigate whether thedeterliorated effect of colonic inflammationon brain I/R injuryis related to the down-regulation of endogenous H2S.3.To clarify whether the neuroprotective effect of CSE-derived H2S against cerebral I/R injury is related to inhibiting the RhoA/ROCK pathway and then promoting the transformation of reactive astrocytes into A2 phenotype.Method:Part I:The mutual promotion of hydrogen sulfide against cerebral ischemia/reperfusion injury and colonic inflammation1.Bilateral common carotid artery ligation（CCA）was used to construct mouse brain I/R model;mice were allowed to drink 3%DSS for seven days to induce colitis model.1.1 Healthy male Kunming mice were randomly divided into 5 groups,10 mice in each group,control group,colitis group,cerebral I/R group,cerebral I/R+colitis group,and cerebral I/R+colitis+Na HS group.1.2 Assessmentof colon damage in mice by observing the survival rate,percentage of body weight loss,colon length and disease activity index（DAI）.1.3 The open field test was used to detect the movement exploration ability of mice.1.4 Hematoxylin-eosin（HE）and Nissl staining were used to observe neuronal damage in hippocampus,and HE staining was used to observe colon damage.1.5 Detection of neuron-specific enolase（NSE）,interleukin-6（IL-6）and tumor necrosis factor-α（IL-6）in mouse serum,testing the TNF-αcontent and H2S content in brain tissue and colon tissue by ELISA.1.6 Detection the expression of L-cystathionine-γ-lyase（CSE）,L-cystathionine-β-synthetase（CBS）,3-mercaptopyruvate sulfurtransferase（3-MST）,Ras’s homologue gene family member A（RhoA）,Rho protein kinase 1（ROCK1）,Rho protein kinase 2（ROCK2）,glial fibrillary acidic protein（GFAP）,S100 calcium binding protein A10（S100A10）and C3 expression by western blot.Part II:CSE-derived H2S promotes the transformation of reactive astrocytes into A2 subtypes to protect against cerebral ischemia-reperfusion injury2.Bilateral common carotid artery ligationwas used to construct cerebral I/R model of wild type（WT）and CSE knockout（knock out,KO）mice.2.1 Experimental groups:Wild-type C57BL/6 and CSE knockout mice were randomly divided into 5 groups,10 mice in each group,WT+sham group,WT+cerebral I/R group,WT+cerebral I/R+Na HS（4.8 mg/kg）group;KO+cerebral I/R group,KO+cerebral I/R+Na HS（4.8 mg/kg）group.Another 30 wild-type C57BL/6 mice were randomly divided into 3 groups:WT+sham group,WT cerebral+I/R group,WT+cerebral I/R+Fasudil（10 mg/kg）group,10 mice in each group.2.2 The open-field test and water maze test were used to test the learning and motor exploration ability of mice.2.3 HE staining was used to observe the damage of neurons in the hippocampus.2.4 Enzyme-linked immunosorbent assay（ELISA）was used to detect the serum neuron-specific enolase（NSE）,lactate dehydrogenase（LDH）content,and test the RhoA protein activity,ROCK1 protein activity,ROCK2 protein activity and the content of H2S in brain tissue lysates.2.5 The expression of RhoA,ROCK1,ROCK2,GFAP,S100A10 and C3 was detected by Western Blot.2.6 The expression of myelin basic protein（MBP）and neuronal nuclear antigen（Neu N）was detected by immunofluorescence method,and the co-expression of Brd U/GFAP,GFAP/S100A10,and GFAP/C3 was detected by immunofluorescence double-labeling method.Result:Part 1:The protective effect of hydrogen sulfide on themutual exacerbation of cerebral ischemia/reperfusion injury and colonic inflammation1.The results of colon length,weight loss percentage and disease activity index showed that weight loss percentage,and disease activity index were reduced after DSS modeling compared with the control group,colon length,indicating that colitis modeling was successful.Compared with the colitis group,the colonic length and body weight decreased,and the DAI scores increased significantly in the cerebral I/R+colitis group.After treatment with Na HS（4.8 mg/kg）,the colonic length and the percentage of body weight loss were reduced,and the DAI scores increased.2.The resultsof open-field testshowed that the mice in the cerebral I/R group had significantly lower total moving distance,moving speed,crossing the line（crossing to adjacent squares）,and standing timescompared with the control group,suggesting that the cerebral I/R group induces behavioral dysfunctionin mice.In contrast,the cerebral I/R+colitis group induced more severe behavioral deficits.Administration of Na HS（4.8 mg/kg）significantly blocked behavioral dysfunction induced by brain I/R and colitis in mice.3.The results of HE staining showed that the histological scores of the colon tissues in the colitis group was higher than that in the control group and the cerebral I/R group.Although cerebral I/R had no effect on the histological scores of the colon in mice,the histological score of the colon in the cerebral I/R+colitis group was higher than that in the colitis group.The results of HE and Nissl staining showed that the histological scores of the brain tissue of the mice in the cerebral I/R group were higher than those in the control group and colitis group.Although the colitis had no effect on the histological scores of the mice brain tissues,the histological scores of the mice brain tissuesin the cerebral I/R+colitis group was higher than that of the mice in the brain I/R group.The supplement with Na HS decreased the histological scores of colon tissue and brain tissues induced by mouse brain I/R and DSS.4.ELISA kit was used to detect the content of inflammatory factors in serum,including IL-6 and TNF-α.The results showed that compared with the control mice,the serum levels of IL-6 and TNF-αinduced by the cerebral I/R group or the colitis group were significantly higher than those of the control mice,indicating that colitis or brain I/R couldinduce inflammation.However,compared with the colitis group and the cerebral I/R group,the cerebral I/R+colitis group induced more significant release of IL-6 and TNF-α（P<0.05）,and Na HS could inhibit the release of these two factors.5.Western blot results showed that compared with the control mice,the expressions of CBS,CSE and 3-MST and the content of H2S in the colon tissue of the colitis mice were significantly decreased,while the expressions of RhoA,ROCK1 and ROCK2 were significantly increased.Cerebral I/R+colitis induced the further decreased expression of endogenous H2S synthase,H2S content in colon tissue,and leaded to the increased expression of RhoA/ROCK pathway proteins（compared with the colitis group,P<0.01）,Na HS couldinhibit the changes of the above protein expression levels.Importantly,there were no significant differences in H2S synthase and RhoA/ROCK pathway protein expression in mouse colon tissue between the control and brain I/R groups.6.In addition,in the hippocampus,we also found that the protein expression of CBS and CSE and the content of H2S were significantly decreased after cerebral I/R,while the expressions of RhoA,ROCK1,and ROCK2 were significantly increased（P<0.05,compared with the control group）.The colitis group also had no significant effect on the expression of endogenous H2S synthase and RhoA/ROCK pathway proteins in the hippocampus;however,cerebral I/R+colitis induced the further reduction of H2S synthaseand H2Scontent in the hippocampus,and blockedthe,and resulted in the increased expression of RhoA/ROCK pathway protein,Na HS couldinhibit the changes of the above protein expression levels.7.The results of western bot showed that compared with the control group,the expression of GFAP in the colon tissue inthe colitis group was decreased,and the expression of C3 protein and S100A10 was down-regulated.In addition,compared with the colitis group,the reduction of GFAP,C3 protein and S100A10 in the colon tissues of cerebral I/R+colitis mice wasmore significant.However,cerebral I/R alone had no effect on GFAP expression in colon tissue.Na HS treatment significantly blocked the reduction of GFAP and S100A10 expression induced by colitis after cerebral I/R in mice,but itfurther reduced the expression of C3 protein.8.In brain tissue,the results showed that brain I/R induced the up-regulation of GFAP,C3 and S100A10 proteins.The up-regulation of GFAP,C3 and S100A10proteins in brain tissue of cerebral I/R+colitis mice were more significant than that of mice in cerebral I/R group.However,colitis had no effect on GFAP expression in the hippocampus.Furthermore,we found that Na HS,exogenous H2S,inhibited the increase of GFAP and C3 expression in the hippocampus;but H2S promoted the expression of S100A10.Part II:CSE-derived H2S regulates A1/A2 astrocyte activation against cerebral ischemia-reperfusion injury1.The results of the open-field testand water maze testshowed that compared with the sham group of WT mice,cerebral I/R could lead to a significant decrease in the motor exploration and learning and memory abilities of WT mice,which was more significant KO mice after cerebral I/Rlessthan that of WT mice.Compared with the cerebral I/R group of CSE KO mice,the motor exploration and learning and memory abilities of CSE KO mice were significantly improved after Na HS treatment.3.The results of HE staining and immunofluorescence staining for Neu N and MBPshowed thatthe WT mice in the I/R group had obvious neuronal damage in the hippocampus of the brain tissues compared with the WT mouse sham group,but the brain injury of KO mice was moreobvious.Compared with the KO micecerebral I/R group,Na HStreatment could significantly improved the brain damageof CSE KO mice.4.ELISA was used to detect the changes of NSE and LDH in mouse serum,and RhoA activity,ROCK1 activity,ROCK2 activity and H2S content in brain tissues.The results showed thatthe above injury factors in the serum and brain tissue of the WT mice in the cerebral I/R group were significantly increased compared with the sham group of WT mice,but the cerebral I/R injury of the CSE KO mice was more significantthan that of the WT mice.Compared with the cerebral I/R group of KO mice,the brain injury of CSE KO mice treated with Na HS was significantly ameliorated.5.The results of western blot showed that the expression of RhoA,ROCK1,ROCK2,GFAP,C3 and S100A10 proteins in the hippocampus of mice after cerebral I/R increased compared with the WT mice sham-operated group.In addition,cerebral I/R-induced expression of RhoA,ROCK1,ROCK2,GFAP,C3 and S100A10 proteins in the brain tissue of CSE KO mice was higher than that of WT mice.However,there was no significant difference in the expression of RhoA,ROCK1,ROCK2,GFAP,C3and S100A10 proteins in the hippocampus of WT sham and CSE KO sham mice.Administration of Na HS inhibited the increase of RhoA,ROCK1,ROCK2,GFAP and C3 expression,but promoted the expression of S100A10.6.The results of double immunofluorescence staining showed the increased number of Brd U/GFAP,GFAP/C3,GFAP/S100A10 co-localized cells in the hippocampus of mice after cerebral I/R（compared with the sham group,P<0.01）.However,the increase of Brd U/GFAP,GFAP/C3 and GFAP/S100A10 positive cells in the hippocampus of WT mice induced by cerebral I/R was significantly higher than that of CSE knockout mice.In the Na HS-treated group,brain I/R-induced Brd U/GFAP and GFAP/C3 co-localized cells in the hippocampus of mice were significantly reduced,while the proliferation of GFAP/S100A10 positive cells in the hippocampus was promoted.7.In the wild-type C57BL/6 mice,compared with the sham group,the motor exploration and learning and memory abilities of the mice in the brain I/R group were significantly reduced,and there was obvious pathological damage in the hippocampus of the brain tissues.At the same time,RhoA,ROCK1,ROCK2,GFAP,C3 and S100A10 protein expressions were significantly increased.After treatment with 10mg/kg ROCK inhibitor Fasudil,the exercise abilityandlimb coordination ability of mice and brain injuryin mice hippocampus were significantly improved,and RhoA,ROCK1,ROCK2,GFAP,C3 and S100A10 expression were significantly decreased.Conclusion:1.Cerebral I/R aggravates the acute intestinal inflammation of mice,meanwhile,colitis following the cerebral I/R exacerbates the brain injury.Mutual exacerbation of colitis and cerebral I/R injury is related to the reduction of endogenous H2S and subsequent up-regulation of RhoA/ROCK pathway.2.The neuroprotective effect of CSE-produced H2S is related to inhibiting the RhoA/ROCK pathwy and then promoting the transformation of reactive astrocytes into A2 subtypes. |
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